Extracellular matrix material conduits and methods of making and using same

Title: Extracellular matrix material conduits and methods of making and using same.Abstract: Extracellular matrix (ECM) material conduits are disclosed. Methods for regenerating atrioventricular valves to replace defective atrioventricular valves within a heart of a subject using the ECM material conduits are also disclosed. Methods of sterilizing and decellularizing an ECM material are also disclosed. ...

The Patent Description & Claims data below is from USPTO Patent Application 20120303117, Extracellular matrix material conduits and methods of making and using same.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of the filing dates of U.S. Provisional Patent Application No. 61/490,693, filed on May 27, 2011, U.S. Provisional Patent Application No. 61/490,873, filed on May 27, 2011, U.S. Provisional Patent Application No. 61/491,723, filed on May 31, 2011, and U.S. Provisional Patent Application No. 61/650,911, filed on May 23, 2012, each of which is hereby incorporated by reference herein in its entirety.

FIELD

The invention relates to extracellular matrix (ECM) material conduits and methods of using such ECM material conduits to regenerate atrioventricular (AV) valves within a heart of a subject.

BACKGROUND

There are many known types of replacement heart valves. The selection of a particular type of replacement heart valve depends on factors such as the location of the valve, the age and other specifics of the patient, and the surgeon's experiences and preferences. Commonly used replacement heart valves can be classified in the following three groups: mechanical valves; allograft tissue valves; and xenograft tissue valves.

Mechanical heart valves, including, for example and without limitation, caged-ball valves, bi-leaflet valves, and tilting disk valves are typically attached to a sewing ring so that the valve prosthesis can be sutured to the patient's native tissue to hold the mechanical valve in place postoperatively. Although mechanical heart valves have advantageous long-term durability, these mechanical valves also have a propensity to cause the formation of blood clots in a patient. If such blood clots form on the mechanical valve, they may preclude the valve from opening or closing correctly or, more importantly, may disengage from the valve and embolize to the brain, causing an embolic stroke. Thus, the patients who receive such mechanical valves are typically required to take systemic anticoagulant drugs for the rest of their lives. In addition to being expensive, these anticoagulant drugs can themselves be dangerous in that they can cause abnormal bleeding in the patient that can lead to a hemorrhagic stroke.

Allograft tissue valves are harvested from human sources, such as human cadavers. Unlike mechanical heart valves, allograft tissue valves typically do not promote blood clot formation and, therefore, avoid the need for prescribing an anticoagulant medication for the patient. However, allograft tissue valves are not available in sufficient numbers to satisfy the needs of all patients who need new heart valves. Furthermore, there have been significant complications when allograft tissue valves have been used to replace atrioventricular (AV) valves within a subject. Moreover, allograft tissue valves can be more difficult to implant than mechanical valves or xenograft valves. Because of these difficulties in implantation, the operative risk associated with allograft tissue valves is often greater than the operative risks associated with mechanical valves and xenograft valves.

Xenograft tissue valves are formed from non-human tissue sources, such as cows or pigs. Most known xenograft tissue valves are constructed by sewing and/or constructing valve leaflets from a non-human tissue source and then securing the leaflets within a patient's heart using a stent and/or a sewing ring. These xenograft tissue valves are less likely to cause blood clot formation than comparable mechanical valves, and therefore, patients that receive xenograft tissue valves are not always required to take anticoagulant medications. However, xenograft tissue valves are prone to calcification and lack the long-term durability of mechanical valves and, consequently, require frequent replacement as compared to mechanical valves. One factor that may contribute to these failures is the chemical treatment that the xenograft tissue valves typically undergo to reduce antigenicity of the animal tissue. Without these chemical treatments, xenograft tissue valves can trigger an immune response in a patient, which can lead to rejection of the tissue valve by the patient. Another factor that may contribute to the lack of durability of the xenograft tissue valves is the presence of a stent and/or sewing ring, which can prevent the xenograft tissue valve from accurately approximating the anatomy of a normal heart valve.

Known tissue conduits, including those described in U.S. Pat. Nos. 5,480,424 and 5,713,950, both of which are expressly incorporated herein by reference in their entirety, suffer from various limitations, including many of the limitations of known xenograft tissue valves. For example, known tissue conduits suffer from antigenicity of the conduits, which is typically addressed using chemical treatments that lessen post-implantation durability of the conduit. Additionally, these known conduits are rapidly degraded within a patient's heart such that they can only serve as competent heart valve replacements for a matter of months.

Thus, what is needed in the art is a readily available, highly durable, and affordable tissue prosthesis that can be easily implanted to regenerate an anatomically accurate AV valve within the heart of a subject. There is a further need in the art for a sterile, acellular tissue prosthesis that can be implanted to regenerate an AV valve within the heart of a subject.

SUMMARY

Extracellular matrix (ECM) material conduits are disclosed. In one aspect, a disclosed ECM material conduit defines a lumen and has an inlet portion and an outlet portion. The inlet portion of the ECM material conduit includes an inlet of the lumen. The outlet portion of the ECM material conduit includes an outlet of the lumen. The inlet portion and the outlet portion of the ECM material conduit can each have an outer circumference. The ECM material conduit is sterile and acellular. Methods of regenerating an atrioventricular (AV) valve to replace a defective AV valve within a heart of a subject are also disclosed. In one aspect, the methods include removing the defective AV valve from the heart of the subject. The methods also include implanting an ECM material conduit within the heart of the subject to regenerate a functional AV valve.

BRIEF DESCRIPTION OF THE FIGURES

These and other features of the preferred embodiments of the invention will become more apparent in the detailed description in which reference is made to the appended drawings wherein:

FIG. 1 is a cut-away view of a human heart.

FIG. 2 is a perspective view of an ECM material conduit as it is attached to an annulus of an atrioventricular valve and to two papillary muscles, as described herein.

FIG. 3 is a bottom perspective view of the ECM material conduit shown in FIG. 2. The ECM material conduit depicted in FIG. 3 is attached to a first papillary muscle at a first attachment point and to a second papillary muscle at a second attachment point.

FIG. 4 is an image of a native tri-cuspid valve following removal from the heart of a subject. FIG. 4 includes markings corresponding to particular measurements that were performed on various native porcine and human tri-cuspid valves.

FIG. 5 is an image of an ECM material conduit following implantation of the ECM material conduit following removal of a native tri-cuspid valve. FIG. 5 displays the ECM material conduit in a closed position within an in vitro model of the right heart.

FIG. 6 depicts Doppler echocardiography images taken postoperatively for an exemplary ECM material conduit functioning as a tri-cuspid valve within an animal. FIG. 6(a) depicts the ECM material conduit one month following the operation with the valve in a closed position. FIG. 6(b) depicts the ECM material conduit immediately post-operatively with the valve in an open position. FIG. 6(c) depicts the ECM material conduit immediately post-operatively with the valve in a closed position.

FIG. 8 displays an image of a regenerated tri-cuspid valve at three months following implantation of an exemplary ECM material conduit as described herein.

FIGS. 9-10 depict the results of an experiment in which DNA content was measured for small intestinal submucosa (SIS) compositions following various sterilization methods, including the sterilization methods described herein. FIG. 9 shows the DNA content of each SIS composition following sterilization. FIG. 10 shows the percentage of DNA that was removed from each SIS composition following sterilization, as compared to raw, unprocessed SIS.

FIGS. 11-12 depict the results of an experiment in which native growth factor content was measured for SIS compositions following various sterilization methods, including the sterilization methods described herein. FIG. 11 shows the bFGF content of each SIS composition (normalized by dry weight of samples) following sterilization. FIG. 12 shows the active TGF-β content of each SIS composition (normalized by dry weight of samples) following sterilization.

FIG. 13 depicts the results of an experiment in which bFGF was incorporated into SIS compositions during rapid depressurization, as described herein. FIG. 13 shows the bFGF content for each SIS composition (normalized by dry weight of samples) following rapid depressurization.

FIG. 14 depicts the results of an experiment in which the tensile strength of two-ply SIS compositions was measured following various sterilization methods, including the sterilization methods described herein. FIG. 14 shows the tensile strength measured for each SIS composition following sterilization.

FIG. 15 depicts the results of an experiment in which native growth factor content was measured for SIS compositions following various sterilization and/or decellularization methods, including the sterilization and decellularization methods described herein. FIG. 15 shows the bFGF enzyme-linked immunosorbent assay (ELISA) results for each SIS composition (normalized by dry weight of samples) following sterilization and/or decellularization.

FIG. 16 shows the DNA content in SIS after it is processed in various ways. The baseline measurement is raw. The tissue was then exposed to supercritical CO2 followed by rapid depressurization (RDP) to facilitate enhanced removal of DNA and cellular debris. After the RDP, the tissue was placed in supercritical CO2 with peracetic acid (PAA) for sterilization. The comparison is to processed SIS either unsterilized or sterilized with ethylene oxide (ETO).

FIG. 17 shows the Percent removal of DNA from SIS after it is processed in various ways. The baseline measurement is raw. The tissue was then exposed to supercritical CO2 followed by rapid depressurization (RDP) to facilitate enhanced removal of DNA and cellular debris. After the RDP, the tissue was placed in supercritical CO2 with peracetic acid (PAA) for sterilization. The comparison is to processed SIS either unsterilized or sterilized with ethylene oxide (ETO).

FIG. 18 shows the variable active Transforming Growth Factor (TGF-beta) content in SIS after it is processed in various ways. The baseline measurement is raw, or unprocessed SIS followed by processing with only Triton X-100 (TX-100) detergent. The tissue was then exposed to supercritical CO2 followed by rapid depressurization (RDP) to facilitate enhanced removal of DNA and cellular debris. After the RDP, the tissue was placed in supercritical CO2 with peracetic acid (PAA) for sterilization. The comparison is to processed SIS either unsterilized or sterilized with ethylene oxide (ETO).

FIG. 19 shows the variable basic Fibroblast Growth Factor (bFGF) content in SIS after it is processed in various ways. The baseline measurement is raw, or unprocessed SIS followed by processing with only Triton X-100 (TX-100) detergent. The tissue was then exposed to supercritical CO2 followed by rapid depressurization (RDP) to facilitate enhanced removal of DNA and cellular debris. After the RDP, the tissue was placed in supercritical CO2 with peracetic acid (PAA) for sterilization. The comparison is to processed SIS either unsterilized or sterilized with ethylene oxide (ETO).

FIG. 20 shows the addition of basic Fibroblast Growth Factor (bFGF) content to SIS using rapid depressurization. The baseline measurement is raw, or unprocessed SIS. The comparison is to processed SIS either unsterilized or sterilized with ethylene oxide (ETO).

DETAILED DESCRIPTION

The present invention may be understood more readily by reference to the following detailed description, examples, and claims, and their previous and following description. However, before the present devices, systems, and/or methods are disclosed and described, it is to be understood that this invention is not limited to the specific devices, systems, and/or methods disclosed unless otherwise specified, as such can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting.

As used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to an “attachment point” can include two or more such attachment points unless the context indicates otherwise.

Ranges may be expressed herein as from “about” one particular value, and/or to “about” another particular value. When such a range is expressed, another aspect includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another aspect. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint.

As used herein, the terms “optional” and “optionally” mean that the subsequently described event or circumstance may or may not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.

The word “or” as used herein means any one member of a particular list and also includes any combination of members of that list.

Unless otherwise expressly stated, it is in no way intended that any method or aspect set forth herein be construed as requiring that its steps be performed in a specific order. Accordingly, where a method claim does not specifically state in the claims or descriptions that the steps are to be limited to a specific order, it is in no way intended that an order be inferred, in any respect. This holds for any possible non-express basis for interpretation, including matters of logic with respect to arrangement of steps or operational flow, plain meaning derived from grammatical organization or punctuation, or the number or type of aspects described in the specification.

Without the use of such exclusive terminology, the term “comprising” in the claims shall allow for the inclusion of any additional element—irrespective of whether a given number of elements is enumerated in the claim, or the addition of a feature could be regarded as transforming the nature of an element set forth in the claims. Except as specifically defined herein, all technical and scientific terms used herein are to be given as broad a commonly understood meaning as possible while maintaining claim validity.

As used herein, a “subject” is an individual and includes, but is not limited to, a mammal (e.g., a human, horse, pig, rabbit, dog, sheep, goat, non-human primate, cow, cat, guinea pig, or rodent), a fish, a bird, a reptile or an amphibian. The term does not denote a particular age or sex. Thus, adult and newborn subjects, as well as fetuses, whether male or female, are intended to be included. A “patient” is a subject afflicted with a disease or disorder. The term “patient” includes human and veterinary subjects. As used herein, the term “subject can be used interchangeably with the term “patient.”

As used herein, the term “circumference” refers to the perimeter of, or the length measurement of the boundary defined by, a closed planar figure. Optionally, as used herein, a “circumference” can correspond to the perimeter of a closed planar circle. However, it is contemplated that a “circumference” can correspond to the perimeter of any closed planar figure, such as, for example and without limitation, an oval, square, rectangular, trapezoidal, or nonsymmetrical closed planar figure. For example, as used herein, an outer “circumference” of a conduit corresponds to the perimeter of the closed planar figure defined by an outer surface of the conduit at a particular location along the longitudinal axis of the conduit.

As used herein, the term “frusto-conical” refers to the shape of a conical frustum, which corresponds to the shape of a cone that has had its tip truncated by a plane parallel to its base. Thus, as used herein, a “frusto-conical” conduit has a substantially circular cross-section that varies in diameter along its longitudinal axis. The “frusto-conical” conduits disclosed herein have inlet portions and outlet portions that each have outer circumferences. Optionally, the outer circumference of the outlet portion of a disclosed “frusto-conical” conduit can be greater than the outer circumference of the inlet portion of the “frusto-conical” conduit. Alternatively, the outer circumference of the outlet portion of a disclosed “frusto-conical” conduit can be less than the outer circumference of the inlet portion of the “frusto-conical” conduit. In exemplary aspects, the outer circumference of the outlet portion of a disclosed “frusto-conical” conduit can be substantially equal to the outer circumference of the inlet portion of the “frusto-conical” conduit.

As used herein, the term “acellular” is meant to describe extracellular matrix compositions that are at least 80% decellularized such that the extracellular matrix composition is at least 80% without cells and/or cellular remnants. In some exemplary aspects described herein, the term “acellular” can refer to extracellular matrix compositions that are at least 90% decellularized such that the extracellular matrix composition is at least 90% without cells and/or cellular remnants. In other exemplary aspects described herein, the term “acellular” can refer to extracellular matrix compositions that are at least 95% decellularized such that the extracellular matrix composition is at least 95% without cells and/or cellular remnants. In other exemplary aspects described herein, the term “acellular” can refer to extracellular matrix compositions that are at least 96% decellularized such that the extracellular matrix composition is at least 96% without cells and/or cellular remnants. In still other exemplary aspects described herein, the term “acellular” can refer to extracellular matrix compositions that are at least 97% decellularized such that the extracellular matrix composition is at least 97% without cells and/or cellular remnants. In further exemplary aspects described herein, the term “acellular” can refer to extracellular matrix compositions that are at least 98% decellularized such that the extracellular matrix composition is at least 98% without cells and/or cellular remnants. In still further exemplary aspects described herein, the term “acellular” can refer to extracellular matrix compositions that are at least 99% decellularized such that the extracellular matrix composition is at least 99% without cells and/or cellular remnants. Thus, as used herein, the term “acellular” can refer to extracellular matrix compositions that are decellularized at levels of 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, and any percentages falling between these values.

As used herein, the term “additive” refers to materials that can be selectively incorporated into the disclosed ECM materials to impart predetermined properties to the sterilized, acellular ECM compositions disclosed herein. Such additives can include, for example and without limitation, growth factors, cytokines, proteoglycans, glycosaminoglycans (GAGs), proteins, peptides, nucleic acids, small molecules, cells and pharmaceutical agents, such as statin drugs, corticosterioids, anti-arrhythmic drugs, nonsteroidal anti-inflammatory drugs, other anti-inflammatory compounds, nanoparticles, and metallic compounds.

As used herein, the term “contemporaneously” refers to the simultaneous and/or overlapping occurrence of events, as well as the sequential occurrence of events within thirty minutes before or after one another. Thus, if a first event occurs, then a second event can be said to have occurred contemporaneously with the first event if it occurred concurrently with the first event or within thirty minutes before or after the first event. For example, if a first method step is performed, then a second method step performed five minutes after the first method step can be said to be performed “contemporaneously” with the first method step. Similarly, if the second method step was performed ten minutes before performance of a third method step, then the second method step can be said to be performed “contemporaneously” with the third method step.

As used herein, the term “supercritical” refers to a fluid state of a material when it is held at or above its critical temperature and critical pressure. When a material is held at or above its critical temperature and critical pressure, then it typically adopts functional properties of both a gas and a liquid and is said to function as a supercritical fluid. Thus, for example, when carbon dioxide is held at or above its critical temperature (31.1° C.) and its critical pressure (1,071 psi), it behaves as a supercritical carbon dioxide fluid and can, for example, exhibit the expansion properties of a gas while having the density of a liquid.

Described herein with reference to FIGS. 1-3 are methods of making and using extracellular matrix (ECM) material conduits. In one aspect, as depicted in FIGS. 2-3, an exemplary ECM material conduit 10 can define a lumen 12 and have an inlet portion 14 and an outlet portion 18. In this aspect, it is contemplated that the inlet portion 14 of the ECM material conduit 10 can comprise an inlet 16 of the lumen 12. It is further contemplated that the outlet portion 18 of the ECM material conduit 10 can comprise an outlet 20 of the lumen 12.

In additional aspects, the inlet portion 14 and the outlet portion 18 of an ECM material conduit 10 can each have an outer circumference. In one aspect, it is contemplated that the outer circumference of the outlet portion 18 of the ECM material conduit 10 can be substantially equal to the outer circumference of the inlet portion 14 of the ECM material conduit. Optionally, in this aspect, the ECM material conduit 10 can be substantially cylindrical. In another aspect, it is contemplated that the outer circumference of the outlet portion 18 of the ECM material conduit 10 can be greater than the outer circumference of the inlet portion 14 of the ECM material conduit. Optionally, in this aspect, the ECM material conduit 10 can be substantially frusto-conical. In a further aspect, it is contemplated that the outer circumference of the outlet portion 18 of the ECM material conduit 10 can be less than the outer circumference of the inlet portion 14 of the ECM material conduit. Optionally, in this aspect, the ECM material conduit 10 can be substantially frusto-conical.

In one aspect, it is contemplated that the ECM material conduit can have a longitudinal axis 24 and a longitudinal length ranging from about 10 mm to about 50 mm. In another aspect, it is contemplated that the outer circumferences of the inlet portion 14 and the outlet portion 18 of the ECM material conduit 10 can each range from about 25 mm to about 190 mm. Thus, it is further contemplated that, at the inlet portion 14 and the outlet portion 18 of the ECM material conduit 10, the lumen 12 of the ECM material conduit can have a diameter ranging from about 8 mm to about 60 mm. In an additional aspect, the ECM material conduit 10 can have a wall 22 having a thickness. In this aspect, it is contemplated that the thickness of the wall 22 of the ECM material conduit 10 can range from about 0.05 mm to about 3.00 mm.

In one exemplary aspect, the outlet portion 18 of the ECM material conduit 10 can comprise one or more extension portions that protrude outwardly from the ECM material conduit 10. It is contemplated that the extension portions can be configured to provide an attachment configuration for the papillary muscles that more closely mimics native functionality. It is further contemplated that the extension portions can be configured to promote fusion between the native papillary muscles attached to the ECM material conduit 10 and the regenerated AV valve formed following implantation of the ECM material conduit as described herein.

In exemplary aspects, when the outlet portion 18 of the ECM material conduit 10 comprises at least one extension portion as described herein, it is contemplated that one or more of the first, second, and third attachment points can be positioned on a corresponding extension portion of the at least one extension portion. In these aspects, it is contemplated that the at least one extension portion can comprise three extension portions, with each of the first, second, and third attachment points being positioned on a respective extension portion.

In exemplary aspects, a disclosed ECM material conduit can comprise any known ECM component or material, including, for example and without limitation, mucosal layers and components, submucosal layers and components, muscularis layers and components, and/or basement membrane layers and components. It is contemplated that a disclosed ECM material conduit can comprise an ECM material obtained from any mammalian tissue source, including, for example and without limitation, stomach tissue (e.g., stomach submucosa (SS)), small intestinal tissue (e.g., small intestinal submucosa (SIS)), large intestinal tissue, bladder tissue (e.g., urinary bladder submucosa (UBS)), liver tissue (e.g., liver basement membrane (LBM)), heart tissue (e.g., pericardium), lung tissue, kidney tissue, pancreatic tissue, prostate tissue, mesothelial tissue, fetal tissue, a placenta, a ureter, veins, arteries, heart valves with or without their attached vessels, tissue surrounding the roots of developing teeth, and tissue surrounding growing bone. It is further contemplated that a disclosed ECM material conduit can comprise an ECM material obtained from ECM components or materials of one or more mammals including, for example and without limitation, humans, cows, pigs, dogs, sheep, cats, horses, rodents, and the like. Thus, it is contemplated that a disclosed ECM material conduit can comprise ECM components or materials from two or more of the same mammalian species, such as, for example and without limitation, two or more cows, two or more pigs, two or more dogs, or two or more sheep. It is further contemplated that a disclosed ECM material conduit can comprise ECM components or materials from two or more different mammalian species, such as, for example and without limitation, a pig and a cow, a pig and a dog, a pig and a sheep, or a cow and a sheep. It is still further contemplated that a disclosed ECM material conduit can comprise ECM components or materials obtained from a first tissue source, such as, for example and without limitation, SIS, from a first mammal, as well as ECM components or materials obtained from a second tissue source, such as, for example and without limitation, SS, from a second mammal.

In various aspects, a disclosed ECM material conduit 10 can be formed from a substantially flat sheet of ECM material. In these aspects, the ECM material conduit 10 can be formed by securing a first edge of the sheet of ECM material to a second, opposed edge of the sheet of ECM material such that a lumen 12 of the-ECM material conduit is defined. It is contemplated that the first edge of the sheet of ECM material can be secured to the second edge of the sheet of ECM material using any conventional surgical attachment means, including, for example and without limitation, non-absorbable sutures, absorbable sutures, surgical pastes, surgical glues, staples, and the like. In one exemplary aspect, when non-absorbable sutures are used to secure the first edge of the sheet of ECM material to the second, opposed edge of the sheet of ECM material, it is contemplated that the non-absorbable sutures can be positioned on an outer surface of the ECM material conduit, thereby reducing the portion of the sutures positioned within the lumen of the ECM material conduit. In one optional aspect, it is contemplated that the second edge of the sheet of ECM material can be secured in overlapping relation with the first edge of the sheet of ECM material. In this aspect, it is further contemplated that the portion of the ECM material conduit 10 at which the first and second edges of the sheet overlap can be everted relative to the lumen of the ECM material conduit. In another optional aspect, it is contemplated that the second edge of the sheet of ECM material can be secured in substantial alignment with the first edge of the sheet of ECM material.

In additional aspects, a disclosed ECM material conduit 10 can comprise at least a portion of an intact, lumenal ECM material, such as, for example and without limitation, a lumenal portion of a native SIS layer. In these aspects, the intact ECM material defines a lumen.

In further aspects, a disclosed ECM material conduit 10 can be formed by growing cells, such as, for example and without limitation, fibroblasts, on an outer surface of a cylindrical mandrel using known in vitro methods. In these aspects, it is contemplated that the growth of cells on the outer surface of the mandrel can lead to production of one or more ECM materials. It is further contemplated that the ECM material conduit 10 can be decellularized using known methods or as disclosed herein.

In an additional aspect, a disclosed ECM material conduit 10 can be lyophilized using known methods. In a further aspect, when a disclosed ECM material conduit 10 has been lyophilized, it is contemplated that the ECM material conduit can be hydrated using known methods. In this aspect, it is contemplated that the lyophilized ECM material conduit can be hydrated in sterile water, saline solution, or a balanced salt solution for a period ranging from about 5 minutes to about 30 minutes.

Optionally, a disclosed ECM material conduit 10 can be a multi-layer construction of two or more layers of ECM material. In one exemplary aspect, a multi-layer ECM material conduit 10 can be formed from a lumenal portion of an intact ECM. As used herein, the term “lumenal” refers to a portion of a material that defines a lumen. In this aspect, the intact lumenal ECM can have a first end and a second end and can define a lumen. Optionally, the first end of the intact ECM can be inverted into the lumen until it reaches the second end, thereby creating a multi-layer ECM material conduit. Alternatively, the first end of the intact SIS can be everted over itself until it reaches the second end, thereby creating a multi-layer ECM material conduit. In a further aspect, the multi-layer ECM material conduit can be lyophilized using known techniques, thereby creating a multi-laminate ECM material conduit. In one optional aspect, the multi-layer ECM material conduit can be positioned over a mandrel during lyophilization. In an alternative, optional aspect, during lyophilization of the multi-layer ECM material conduit, a cryoballoon can be positioned within the lumen of the multi-layer ECM material conduit and then inflated to press together the layers of the multi-layer ECM material conduit. It is further contemplated that any conventional lamination method can be used to laminate the layers of a multi-layer ECM material conduit together, thereby forming a multi-laminate ECM material conduit.

In one aspect, a disclosed ECM material conduit 10 can comprise a sterile, acellular ECM composition. In exemplary aspects, such a sterile, acellular ECM composition can be formed by contemporaneously sterilizing and decellularizing an isolated ECM material. More particularly, as disclosed in the following methods, desired sterilization and decellularization of the isolated ECM material can occur contemporaneously such that the native properties of the tissue composition are maintained and the ECM material is rendered sterile and acellular.

In exemplary aspects, the ECM material conduit 10 can have a multi-layer structure proximate the inlet and/or outlet portion of the ECM material conduit. In these aspects, it is contemplated that at least one end of the ECM material conduit can be everted or inverted along a portion of the length of the ECM material conduit to thereby form a multi-layer structure proximate the inlet and/or outlet portion of the ECM material conduit. It is further contemplated that the multi-layer structure can effectively act as a sewing ring for the ECM material conduit.

Sterilization and Decellularization of ECM Compositions for Use in ECM Material Conduits

As described herein, the disclosed methods make use of rapid depressurization of an isolated ECM material to decellularize the ECM material such that it is acellular. This rapid depressurization of the ECM material occurs at depressurization rates that are significantly higher than the depressurization rates applied in previously known methods. In addition to decellularizing the ECM material as described herein, the rapid depressurization of the ECM material also can be used to incorporate desired sterilants and additives into the ECM material.

Optionally, it is contemplated that the ECM material of a disclosed ECM valve conduit can be sterilized using a known sterilization system, such as, for example and without limitation, the system described in U.S. Pat. No. 7,108,832, assigned to NovaSterilis, Inc., which patent is expressly incorporated herein by reference in its entirety. Thus, in some aspects, the system used to perform the disclosed methods can comprise a standard compressed storage cylinder and a standard air compressor used in operative association with a booster (e.g., a Haskel Booster AGT 7/30). In other aspects, the air compressor and booster can be replaced with a single compressor. In exemplary aspects, the compressed storage cylinder can be configured to receive carbon dioxide, and the booster can be a carbon dioxide booster.

The system can further comprise an inlet port, which allows one or more additives contained in a reservoir to be added to a reactor vessel through a valve and an additive line. As used herein, the term “reactor vessel” refers to any container having an interior space that is configured to receive an ECM material and permit exposure of the ECM material to one or more sterilants and additives, as disclosed herein. In exemplary aspects, the reactor vessel can be, without limitation, a basket, a bucket, a barrel, a box, a pouch, and other known containers. In one aspect, it is contemplated that the reactor vessel can be a syringe that is filled with an ECM material.

It is contemplated that a selected primary sterilant, such as, for example and without limitation, carbon dioxide, can be introduced to the reactor vessel from a header line via a valve and a supply line. It is further contemplated that a filter, such as, for example and without limitation, a 0.5 μm filter, can be provided in the supply line to prevent escape of material from the vessel. In exemplary aspects, a pressure gauge can be provided downstream of a shut-off valve in the header line to allow the pressure to be visually monitored. A check valve can be provided in the header line upstream of the valve to prevent reverse fluid flow into the booster. In order to prevent an overpressure condition existing in the header line, a pressure relief valve can optionally be provided.

In one aspect, depressurization of the reactor vessel can be accomplished using an outlet line and a valve in communication with the reactor vessel. In this aspect, it is contemplated that the depressurized fluid can exit the vessel via the supply line, be filtered by a filter unit, and then be directed to a separator, where filtered fluid, such as carbon dioxide, can be exhausted via an exhaust line. It is further contemplated that valves can be incorporated into the various lines of the apparatus to permit fluid isolation of upstream components.

In one exemplary aspect, the reactor vessel can comprise stainless steel, such as, for example and without limitation, 316 gauge stainless steel. In another exemplary aspect, the reactor vessel can have a total internal volume sufficient to accommodate the materials being sterilized, either on a laboratory or commercial scale. For example, it is contemplated that the reactor vessel can have a length of about 8 inches, an inner diameter of about 2.5 inches, and an internal volume of about 600 mL. In additional aspects, the reactor vessel can comprise a vibrator, a temperature control unit, and a mechanical stirring system comprising an impeller and a magnetic driver. In one optional aspect, it is contemplated that the reactor vessel can contain a basket comprising 316 gauge stainless steel. In this aspect, it is contemplated that the basket can be configured to hold materials to be sterilized while also protecting the impeller and directing the primary sterilant in a predetermined manner.

It is contemplated that the reactor vessel can be operated at a constant pressure or under continual pressurization and depressurization (pressure cycling) conditions without material losses due to splashing or turbulence, and without contamination of pressure lines via back-diffusion. It is further contemplated that the valves within the system can permit easy isolation and removal of the reactor vessel from the other components of the system. In one aspect, the top of the reactor vessel can be removed when depressurized to allow access to the interior space of the reactor vessel.

Optionally, the system can comprise a temperature control unit that permits a user to adjustably control the temperature within the reactor vessel.

In use, the disclosed apparatus can be employed in a method of producing a sterilized, acellular ECM composition, such as disclosed herein. However, it is understood that the disclosed apparatus is merely exemplary, and that any apparatus capable of performing the disclosed method steps can be employed to produce the sterilized, acellular ECM composition. Thus, the claimed method is in no way limited to a particular apparatus.

It is contemplated that significant reductions in colony-forming units (CFUs) can be achieved in accordance with the disclosed methods by subjecting an isolated ECM material to sterilization temperature and pressure conditions using a primary sterilant. Optionally, it is contemplated that the primary sterilant can be combined with one or more secondary sterilants to achieve desired sterilization. Optionally, it is further contemplated that selected additives can be incorporated into an ECM material to impart desired characteristics to the resulting ECM composition. It is still further contemplated that the disclosed methods can be employed to produce sterilized, acellular ECM compositions for implantation within the body of a subject.

As described herein, the disclosed methods make use of rapid depressurization of an isolated ECM material to render the ECM material acellular. This rapid depressurization of the ECM material occurs at depressurization rates that are significantly higher than the depressurization rates applied in previously known methods. In addition to rendering acellular the ECM material as described herein, the rapid depressurization of the ECM material also can be used to enhance the incorporation of desired sterilants and additives into the ECM material. Further, it is contemplated that the rapid depressurization of the ECM material can render the ECM material acellular while also improving retention of native growth factors, as compared to previously known decellularization methods. Still further, it is contemplated that the rapid depressurization of the ECM material can be used to improve retention of the tensile strength of the ECM material, as compared to previously known decellularization methods.

The disclosed methods not only do not significantly weaken the mechanical strength and bioptric properties of the ECM compositions, but also the methods are more effective in decellularizing the ECM compositions and in enhancing the incorporation of various additives into the ECM compositions. Thus, the disclosed sterilization and decellularization methods provide ECM compositions that are more decellularized and have a greater capacity to incorporate and then deliver more additives than ECM compositions known in the art. Moreover, the disclosed sterilization and decellularization methods provide ECM compositions that have greater amounts and/or concentrations of retained native growth factors and that have greater tensile strength than sterilized and decellularized ECM compositions known in the art.

In exemplary aspects, the primary sterilant can be carbon dioxide at or near its supercritical pressure and temperature conditions. However, it is contemplated that any conventional sterilant, including, for example, gas, liquid, or powder sterilants that will not interfere with the native properties of the ECM material, can be used as the primary sterilant.

In one exemplary aspect, the disclosed sterilization process can be practiced using carbon dioxide as a primary sterilant at pressures ranging from about 1,000 to about 3,500 psi and at temperatures ranging from about 25° C. to about 60° C. More preferably, when supercritical carbon dioxide is used, it is contemplated that the sterilization process can use carbon dioxide as a primary sterilant at pressures at or above 1,071 psi and at temperatures at or above 31.1° C. In this aspect, the ECM material to be sterilized can be subjected to carbon dioxide at or near such pressure and temperature conditions for times ranging from about 10 minutes to about 24 hours, more preferably from about 15 minutes to about 18 hours, and most preferably, from about 20 minutes to about 12 hours. Preferably, the carbon dioxide employed in the disclosed systems and methods can be pure or, alternatively, contain only trace amounts of other gases that do not impair the sterilization properties of the carbon dioxide. For ease of further discussion below, the term “supercritical carbon dioxide” will be used, but it will be understood that such a term is non-limiting in that carbon dioxide within the pressure and temperature ranges as noted above can be employed satisfactorily in the practice of the disclosed methods. Within the disclosed pressure and temperature ranges, it is contemplated that the carbon dioxide can be presented to the ECM material in a gas, liquid, fluid or plasma form.

The secondary sterilants employed in the disclosed methods can, in some aspects, include chemical sterilants, such as, for example and without limitation, peroxides and/or carboxylic acids. Preferred carboxylic acids include alkanecarboxylic acids and/or alkanepercarboxylic acids, each of which can optionally be substituted at the alpha carbon with one or more electron-withdrawing substituents, such as halogen, oxygen and nitrogen groups. Exemplary species of chemical sterilants employed in the practice of the disclosed methods include, for example and without limitation, hydrogen peroxide (H2O2), acetic acid (AcA), peracetic acid (PAA), trifluoroacetic acid (TFA), and mixtures thereof. In one exemplary aspect, the chemical sterilants can include Sporeclenz® sterilant, which is a mixture comprising acetic acid, hydrogen peroxide, and peracetic acid.

It is contemplated that the secondary sterilants can be employed in a sterilization-enhancing effective amount of at least about 0.001 vol. % and greater, based on the total volume of the primary sterilant. It is further contemplated that the amount of secondary sterilant can be dependent upon the particular secondary sterilant that is employed. Thus, for example, it is contemplated that peracetic acid can be present in relatively small amounts of about 0.005 vol. % and greater, while acetic acid can be employed in amounts of about 1.0 vol. % and greater. Thus, it is contemplated that the concentration of the secondary sterilants can range from about 0.001 vol. % to about 2.0 vol. % and can typically be used as disclosed herein to achieve a sterilization-enhancing effect in combination with the disclosed primary sterilants, such as, for example and without limitation, supercritical carbon dioxide.

In one aspect, the method of producing a sterilized, acellular ECM composition can comprise harvesting a selected tissue from a mammal and rinsing the selected tissue in sterile saline or other biocompatible liquid, including, for example and without limitation, Ringer\'s solution or a balanced biological salt solution. In this aspect, the selected tissue can be, for example and without limitation, stomach tissue (e.g., stomach submucosa (SS)), small intestinal tissue (e.g., small intestinal submucosa (SIS)), large intestinal tissue, bladder tissue (e.g., urinary bladder submucosa (UBS)), liver tissue (e.g., liver basement membrane (LBM)), heart tissue (e.g., pericardium, epicardium, endocardium, myocardium), lung tissue, kidney tissue, pancreatic tissue, prostate tissue, mesothelial tissue, fetal tissue, a placenta, a ureter, veins, arteries, heart valves with or without their attached vessels, tissue surrounding the roots of developing teeth, and tissue surrounding growing bone. In another aspect, the method can comprise freezing the selected tissue for a period ranging from about 12 to about 36 hours, more preferably, from about 18 to about 30 hours, and most preferably, from about 22 to about 26 hours. For example, it is contemplated that the period during which the selected tissue is frozen can be 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours. 24 hours, 25 hours, 26 hours, 27 hours, 28 hours, 29 hours, 30 hours, 31 hours, 32 hours, 33 hours, 34 hours, 35 hours, 36 hours, and any other period of time falling between the preceding values. In an additional aspect, the method can comprise thawing the selected tissue in cold hypotonic tris buffer. Optionally, in this aspect, the method can comprise thawing the selected tissue in cold hypotonic tris buffer on ice with 5 mM ethylenediaminetetraacetic acid (EDTA). In exemplary aspects, it is contemplated that the steps of freezing and thawing the selected tissue can be cyclically repeated up to six times.

In another aspect, the method can comprise isolating an ECM material from the selected tissue. In this aspect, the ECM material can be any material comprising known extracellular matrix components, including, for example and without limitation, stomach tissue (e.g., stomach submucosa (SS)), small intestinal tissue (e.g., small intestinal submucosa (SIS)), large intestinal tissue, bladder tissue (e.g., urinary bladder submucosa (UBS)), liver tissue (e.g., liver basement membrane (LBM)), heart tissue (e.g., pericardium, epicardium, endocardium, myocardium), lung tissue, kidney tissue, pancreatic tissue, prostate tissue, mesothelial tissue, fetal tissue, a placenta, a ureter, veins, arteries, heart valves with or without their attached vessels, tissue surrounding the roots of developing teeth, and tissue surrounding growing bone, and the like. In one exemplary, non-limiting aspect, the step of isolating an ECM material can comprise isolating SIS from a mammalian tissue source. In this aspect, the method can comprise: incising a wall of a small intestine along a path that is substantially parallel to the longitudinal axis of the small intestine; opening the small intestine along the path of the incision such that the small intestine lies flat on a surface; rinsing the small intestine with sterile saline or other biocompatible fluid; mechanically stripping the SIS of the small intestine from the surrounding smooth muscle and serosal layers and from the tunica mucosa, leaving essentially the submucosal and basement membrane layers. However, it is contemplated that the ECM material can be isolated using any conventional technique, including those described in: U.S. Pat. No. 4,902,508; U.S. Pat. No. 5,275,826; U.S. Pat. No. 5,281,422; U.S. Pat. No. 5,554,389; U.S. Pat. No. 6,579,538; U.S. Pat. No. 6,933,326; U.S. Pat. No. 7,033,611; Voytik-Harbin et al., “Identification of Extractable Growth Factors from Small Intestinal Submucosa,” J. Cell. Biochem., Vol. 67, pp. 478-491 (1997); Hodde et al., “Virus Safety of a Porcine-Derived Medical Device: Evaluation of a Viral Inactivation Method,” Biotech. & Bioeng., Vol. 79, No. 2, pp. 211-216 (2001); Badylak et al., “The Extracellular Matrix as a Scaffold for Tissue Reconstruction,” Cell & Developmental Biology, Vol. 13, pp. 377-383 (2002); Robinson et al., “Extracelular Matrix Scaffold for Cardiac Repair,” Circulation, Vol. 112, pp. I-135-I-143 (2005); Hodde et al., “Effects of Sterilization on an Extracellular Matrix Scaffold: Part I. Composition and Matrix Architecture,” J. Mater. Sci.: Mater. Med., Vol. 18, pp. 537-543 (2007); and Hodde et al., “Effects of Sterilization on an Extracellular Matrix Scaffold: Part II. Bioactivity and Matrix Interaction,” J. Mater. Sci.: Mater. Med., Vol. 18, pp. 545-550 (2007), each of which is expressly incorporated herein by reference in its entirety.

In an additional aspect, the method can comprise incubating the isolated ECM material for 24 to 48 hours in 0.5-1% Triton X-100/0.5-1% Deoxycholic acid with 5 mM EDTA in Dulbecco\'s Phosphate Buffered Saline (DPBS) (Lonza Walkersville, Inc.). In this aspect, it is contemplated that flat or sheet-like ECM materials, such as stomach submucosa (SS), small intestinal submucosa (SIS), and bladder submucosa (UBS), can be incubated in a stretched configuration. It is further contemplated that ECM material conduits or other lumenal ECM materials, such as ureters, arteries, veins, and tubular SIS, can be perfused with the various disclosed solutions through soaking and by use of a peristaltic pump.

In a further aspect, after incubation, the method can comprise rinsing the ECM material with DPBS. In this aspect, it is contemplated that the step of rinsing the ECM material can comprise rinsing the ECM material up to six times, including one, two, three, four, five, or six times, with each rinse lasting for about thirty minutes. In an exemplary aspect, it is contemplated that the step of rinsing the ECM material can comprise rinsing the ECM material three times, with each rinse lasting for about thirty minutes.

Optionally, in exemplary aspects, the method can further comprise a second incubation procedure. In these aspects, the second incubation procedure can comprise incubating the ECM material in isotonic tris buffer containing 10-50 μg/mL of RNAase/0.2-0.5 μg/mL DNAase with 5 mM EDTA. It is contemplated that the step of incubating the ECM material in isotonic tris buffer can be performed at a temperature of about 37° C., substantially corresponding to the temperature of a human body. It is further contemplated that the step of incubating the ECM material in isotonic tris buffer can be performed for a period ranging from about 30 minutes to about 24 hours, more preferably, from about 1 hour to about 18 hours, and most preferably, from about 2 hours to about 12 hours. In an additional aspect, the second incubation procedure can further comprise rinsing the ECM material with DPBS. In this aspect, it is contemplated that the step of rinsing the ECM material can comprise rinsing the ECM material three times, with each rinse lasting for about thirty minutes.

In yet another aspect, whether or not the second incubation procedure is performed, the method can comprise storing the ECM material at a temperature ranging from about 1° C. to about 10° C., more preferably, from about 2° C. to about 6° C., and, most preferably, from about 3° C. to about 5° C. In an exemplary aspect, the ECM material can be stored at 4° C.

In an additional aspect, the method can comprise introducing the ECM material into the interior space of the reactor vessel. Optionally, in this aspect, one or more secondary sterilants from the reservoir can be added into the interior space of the reactor vessel along with the ECM material. In these aspects, it is contemplated that the one or more secondary sterilants from the reservoir can be added into the interior space of the reactor vessel before, after, or contemporaneously with the ECM material. It is further contemplated that the temperature control unit can be selectively adjusted to produce a desired temperature within the interior space of the reactor vessel. In a further aspect, the method can comprise equilibrating the pressure within the reactor vessel and the pressure within the storage cylinder. For example, in this aspect, it is contemplated that the pressure within the reactor vessel and the pressure within the storage cylinder can be substantially equal to atmospheric pressure. In yet another aspect, after equilibration of the pressures within the apparatus, the method can comprise operating the magnetic driver to activate the impeller of the reactor vessel. In still a further aspect, the method can comprise selectively introducing the primary sterilant from the storage cylinder into the reactor vessel until a desired pressure within the reactor vessel is achieved. In this aspect, it is contemplated that the step of selectively introducing the primary sterilant into the reactor vessel can comprise selectively activating the air compressor and the booster to increase flow of the primary sterilant into the reactor vessel. In exemplary aspects, the air compressor and booster can be activated to subject the ECM material to supercritical pressures and temperatures, such as, for example and without limitation, the pressures and temperatures necessary to produce supercritical carbon dioxide, for a time period ranging from about 20 minutes to about 60 minutes.

In a further aspect, the method can comprise rapidly depressurizing the reactor vessel. In this aspect, a predetermined amount of primary sterilant, such as, for example and without limitation, supercritical carbon dioxide, can be released from the reactor vessel through the depressurization line. It is contemplated that the primary sterilant can be released from the reactor vessel through opening of the valve coupled to the reactor vessel to thereby rapidly reduce the pressure within the reactor vessel. As used herein, the term “rapid depressurization” refers to depressurization of the reactor vessel at a rate greater than or equal to 400 psi/min. For example, it is contemplated that the reactor vessel can be rapidly depressurized at a depressurization rate ranging from about 2.9 MPa/min. to about 18.0 MPa/min. (about 400 psi/min. to about 2,600 psi/min.), more preferably, from about 5.0 MPa/min. to about 10.0 MPa/min. (700 psi/min. to about 1,500 psi/min.), and, most preferably, from about 7.0 MPa/min. to about 8.0 MPa/min. (about 1,000 psi/min. to about 1,200 psi/min.). Thus, these rapid depressurizations are significantly greater than the 300 psi/min. depressurization rate disclosed in U.S. Pat. No. 7,108,832. Without being bound by any particular theory, it is believed that the disclosed rapid depressurization rates increase the level of decellularization achieved in the ECM material. For example, it is contemplated that the rapid depressurization of a disclosed ECM material can lead to levels of decellularization in the ECM material of greater than about 96%, including 96.1%, 96.2%, 96.3%, 96.4%, 96.5%, 96.6%, 96.7%, 96.8%, 96.9%, 97.0%, 97.1%, 97.2%, 97.3%, 97.4%, 97.5%, 97.6%, 97.7%, 97.8%, 97.9%, 98.0%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, and 100%.

In exemplary aspects, the method can further comprise the step of incorporating one or more additives into the ECM material. In these aspects, it is contemplated that the one or more additives can be provided in either a powder or a liquid form. In one optional aspect, the step of incorporating the one or more additives can comprise introducing the one or more additives into the reactor vessel during the step of rapidly depressurizing the reactor vessel. In this aspect, it is contemplated that the introduction of the one or more additives can be characterized as a conventional foaming process. In another optional aspect, the step of incorporating the one or more additives can comprise introducing the one or more additives into the reactor vessel after the step of rapidly depressurizing the reactor vessel. In this aspect, it is contemplated that the one or more additives can be added to the ECM material after the rapid depressurization of the reactor vessel has caused the ECM material to swell and/or expand, thereby permitting improved penetration of the additives into the ECM material. It is further contemplated that, in an exemplary aspect, the one or more additives can be added to the ECM material within about thirty minutes after the rapid depressurization of the reactor vessel. In a further optional aspect, the step of incorporating the one or more additives can comprise introducing the one or more additives into the reactor vessel both during and after the step of rapidly depressurizing the reactor vessel. In this aspect, it is contemplated that the one or more additives can be released into the reactor vessel in both a quick manner and a slow, extended manner. In still a further optional aspect, the step of incorporating the one or more additives can comprise introducing the one or more additives into the reactor vessel before the step of rapidly depressurizing the reactor vessel.

The disclosed additives can be incorporated into the ECM material to impart selected properties to the resulting sterilized, acellular ECM composition. Thus, it is contemplated that the one or more additives can be selected to replace or supplement components of the ECM material that are lost during processing of the ECM material as described herein. For example, and as described below, the one or more additives can comprise growth factors, cytokines, proteoglycans, glycosaminoglycans (GAGs), proteins, peptides, nucleic acids, small molecules, drugs, or cells. It is further contemplated that the one or more additives can be selected to incorporate non-native components into the ECM material. For example, the one or more additives can comprise, for example and without limitation, growth factors for recruiting stem cells, angiogenic cytokines, and anti-inflammatory cytokines. It is still further contemplated that the one or more additives can be pharmaceutical agents, such as statins, corticosteroids, non-steroidal anti-inflammatory drugs, anti-inflammatory compounds, anti-arrhythmic agents, and the like. It is still further contemplated that the one or more additives can be nanoparticles, such as, for example and without limitation, silver nanoparticles, gold nanoparticles, platinum nanoparticles, iridium nanoparticles, rhodium nanoparticles, palladium nanoparticles, copper nanoparticles, zinc nanoparticles, and other metallic nanoparticles. It is still further contemplated that the one or more additives can be metallic compounds. In one exemplary aspect, the one or more additives can be selected to pharmaceutically suppress the immune response of a subject following implantation of the resulting ECM composition into the body of a subject.

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